CN107843730A - Make the method for the reversible dyeing of target cell - Google Patents
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Abstract
The present invention relates to the method for making the reversible dyeing of target cell.The method of the target cell group defined the invention further relates to separation target cell or by least one common specific acceptor molecule be present.Present invention also offers available for the kit for implementing the method for the invention.
Description
The cross reference of related application
The U.S. Provisional Patent Application that patent application claims are submitted on July 18th, 2011 to United States Patent and Trademark Office
61/508,943 " rights and interests of Method of Reversibly Staining a Target Cell " priority, Shen
Please it is incorporated by by reference herein.
Invention field
The present invention relates to the method for making the reversible dyeing of target cell.It is at least one common special by existing the invention further relates to separation
The target cell of specific receptor molecular definition or the method for target cell group.Present invention also offers available for the method for implementing the present invention
Kit.
Background of invention
Treatment of the cell therapy to many diseases is efficiently to be proven.For example, primary immunodeficiency can be with
Cured by HSCT (HSCT), some leukaemia can pass through allosome HSCT and supplied oil layer
(DLI) combination obtains complete incidence graph (Kolb, H.J. etc., Donor leukocyte transfusions for
treatment of recurrent chronic myelogenous leukemia in marrow transplant
patients.Blood 76(12),2462-2465(1990)).In some clinical settings, virus specific t cell is adopted
Shift the complication to rebuilding immuuoeorapromised host confrontation life-threatening as caused by cytomegalovirus (CMV)
(Riddell, S.R. etc., Restoration of viral immunity in immunodeficient humans by
The adoptive transfer of T cell clones.Science 257 (5067), 238-241 (1992)) or by
Lymphoproliferative disease (Rooney, C.M. etc., the Use of gene- of Epstein-Barr- viruses (EBV) mediation
modified virus-specific T lymphocytes to control Epstein-Barr-virus-related
Lymphoproliferation.Lancet 345 (8941), 9-13 (1995)) it is highly effective.Equally, either from itself
The tumour antigen orientation T cell that tumor infiltrating lymphocyte is still cultivated or designed in vitro is all promising to improving therapy
Candidate item.
Regardless of these interesting clinical observation results, cell therapy is transferred to clinical practice and still needed more extensively
Treat.This mainly due to the fact that, most programs of the cellular preparations for producing immunization therapy are very time-consuming, expenses
Power and costliness.Furthermore it is known that be used for adjust the cell mass of clinical efficacy and usually require to be enriched to very high purity, because
Harmful and threat to life sometimes side effect (such as graft versus host disease(GVH disease) may be mediated for the pollution of " unwanted " cell
(GvHD) mediated by Xenoreactivity T cell in Allogeneic stem cell is transplanted and/or DLI is treated).Study
Show, very small amount adoptive transfer T cell can promote beneficial clinical efficacy, and same rule is also applied for cell mass mediation
Negative effect.Therefore it provides suitable for treatment well-defined high-purity cellular preparations turn into make these promising treatments
Method more effectively and key that is predictable and reducing potential side effect risk.
At present surface marker mediation clinical cytology purifying method often rely on single parameter (e.g., CD34,
MHC polymers).But for most cell masses-no matter be directly derived from it is in vitro or in vitro after cell culture-
The combination of different surfaces label is necessary, really to come out these cellular compartments from other cells.It is for example, natural
Existing regulatory T cells (Treg) represent a kind of promising cell subsets, and it can prevent to be based on allogene HSCT 4
Acute GvHD or autoimmune disease development (Riley, J.L., June, C.H., ＆Blazar, B.R., Human T
regulatory cell therapy:take a billion or so and call me in the
morning.Immunity 30(5),656-665(2009),Randolph,D.A.&Fathman,C.G.,Cd4+Cd25+
regulatory T cells and their therapeutic potential.Annu Rev Med 57,381-402
(2006))。
Except the CD4 expression shared by a large amount of cells, the high-affinity IL- of other labels such as its constitutive expression
2 receptor alpha chains (CD25) need further to reduce heterogeneity.However, CD25 is also expressed on significant portion of non-regulated cell,
The cell includes effector cell and the memory T cell activated recently.Therefore, it has been suggested that use the staining pattern of combination, such as
CD4, CD25, CD127 and CD45RA combination (Miyara, M. etc., Functional delineation and
differentiation dynamics of human CD4+ T cells expressing the
FoxP3transcription factor.Immunity 30 (6), 899-911 (2009), Hoffmann, P. etc., Only the
CD45RA+subpopulation of CD4+CD25high T cells gives rise to homogeneous
regulatory T-cell lines upon in vitro expansion.Blood 108(13),4260-4267
(2006) this and clinically relevant T cell subgroup are more accurately identified).
Current all available cell separation technologies based on clinical marker thing use paramagnetic beads, and the pearl will be marked
The cell mass of note is retained in magnetic field.Thus, highest purity is obtained using the positive enrichment strategy of the target cell group directly marked.
But be still extremely difficult to via the purifying of the combination of several different labels, although difference of the separation with highest purity is thin
Born of the same parents group is required.In addition, after positive-selecting, mark and pearl are usually remained in cellular products, potentially manipulate separation
Cell mass negatively influences its function/viability and such as passes through receptor block.Particularly clinical cytology sorting in, reservation it is thin
Born of the same parents' mark can cause the great applicability insufficiency of accommodation of cell products in patients.It is the problem of in order to evade positive-selecting, many
Clinical cytology processing routine has been changed to exhaust and set.Unfortunately, often very low and depletion method often needs the purity of target cell
The complex mixture of different antibodies is wanted, this allows for its production and application is laborious and expensive.
Therefore, there is still a need for providing a kind of method for being used for cell purification or separation, this method is for example after cell sorting
Allow the release from the cell mass of purifying and remove all components and coloring agent that acceptor combines completely.
Summary of the invention
One aspect of the present invention provides a kind of method for making the reversible dyeing of target cell using detectable label, the target cell bag
Containing acceptor molecule in its surface, methods described includes：
The cell mixture comprising the target cell is set to be contacted with following substances：
(i) receptor binding agent, the receptor binding agent includes at least one (any) binding site B, wherein combining
Site B is specifically bound to the acceptor molecule, wherein the receptor binding agent is via binding site B and the acceptor molecule
Between combination dissociation rate constant (Koff) value be about 0.5 × 10-4sec-1Or it is bigger, the receptor binding agent enters one
Step includes at least one binding partners C, wherein the binding partners C can (reversible) be connected to the combination of polymerisation agent
Site Z,
(ii) polymerisation agent, the polymerisation agent include the reversible of the binding partners C for receptor binding agent
With reference at least two binding site Z, wherein receptor binding agent (i) and polymerisation agent (ii) form that to be bound to the target thin
(multiple) multivalence combination compound of born of the same parents, each multivalence combination compound include and are bound to a polymerisation agent at least
Two receptor binding agents, the multivalence combination compound provide increased affinity relative to the receptor binding agent
Power；With
(iii) detectable label of the multivalence combination compound is bound to,
Wherein described target cell is dyed by the combination of the multivalence combination compound and the target cell, and is based on institute
State the broken of the combination between the binding partners C of receptor binding agent and the binding site Z of the polymerisation agent
Split, the dyeing of the target cell is reversible.
The second aspect of the invention provide this decoration method be used for separate by (at least one common specific) acceptor be present
The purposes of the target cell group of molecular definition.
Another aspect of the present invention provides the kit for making the reversible dyeing (separation) of target cell using detectable label, institute
State the acceptor molecule that target cell is included in its surface.This kit includes：
(i) at least one receptor binding agent, at least one receptor binding agent are described comprising being specifically bound to
(any) binding site B of acceptor molecule, wherein the receptor binding agent is via binding site B and the receptor binding molecule
Between combination dissociation rate constant (Koff) value be about 0.5 × 10-4sec-1Or it is bigger, and the receptor binding agent
At least one binding partners C is further included, wherein the binding partners C can be by the binding site Z of polymerisation agent
It is (reversible) to combine,
(ii) at least one polymerisation agent, at least one polymerisation agent include the institute for receptor binding agent
State binding partners C at least two binding site Z, wherein binding partners C and the polymerisation agent binding site Z energy
Reversible keying is enough formed,
(iii) detectable label, the detectable label be bound to or can be bound to receptor binding agent (i) and/or
Polymerisation agent (ii).
Another aspect of the invention, which provides a kind of receptor binding agent for being specifically bound to acceptor molecule, makes target cell
Purposes in the method for reversible dyeing, the receptor binding agent includes at least one binding site B, wherein the binding site
B is specifically bound to the acceptor molecule, wherein the receptor binding agent is between binding site B and the acceptor molecule
Combination dissociation rate constant (Koff) value be about 0.5 × 10-4sec-1It is or bigger.
An additional aspect of the present invention provides a kind of method for making the reversible dyeing of target cell using detectable label, the target
Cell is included in (at least one) acceptor molecule on its surface, and methods described includes：
Make contact of the cell mixture comprising the target cell with following substances：
(i) at least two receptor binding agent, every kind of receptor binding agent includes at least one binding site B, wherein often
The binding site B of kind receptor binding agent is specifically bound to the acceptor molecule, wherein every kind of receptor binding agent warp
By the dissociation rate constant (K of the combination between binding site B and the acceptor moleculeoff) value be about 0.5 × 10-4sec-1Or
Bigger, every kind of receptor binding agent further includes at least one binding partners C, wherein every kind of receptor binding agent
Binding partners C can (reversible) be bound to the binding site Z of polymerisation agent,
(ii) polymerisation agent, the polymerisation agent include the binding partners C's for every kind of receptor binding agent
At least one binding site Z of Reversible binding,
Wherein described at least two receptor binding agent (i) and polymerisation agent (ii), which are formed, is bound to the target cell
(multiple) multivalence combination compound, each multivalence combination compound include the every kind of acceptor for being bound to a polymerisation agent
At least one in binding reagents, the multivalence combination compound provides increased parent relative to every kind of receptor binding agent
With joint efforts；With
(iii) detectable label of the multivalence combination compound is bound to,
Wherein described target cell is dyed by the combination of the multivalence combination compound and the target cell, and wherein base
Between the binding partners C of every kind of receptor binding agent and the binding site Z of the polymerisation agent
With reference to division, the dyeing of the target cell is reversible.
Another aspect of the present invention provides the kit for making the reversible dyeing of target cell (or separation) using detectable label,
The target cell includes acceptor molecule in its surface, and the kit includes：
(i) at least two receptor binding agent, at least two receptor binding agent are described comprising being specifically bound to
The binding site B of acceptor molecule, wherein every kind of receptor binding agent via binding site B and the receptor binding molecule it
Between combination dissociation rate constant (Koff) value be about 0.5 × 10-4sec-1Or it is bigger, and every kind of acceptor combines examination
Agent further includes at least one binding partners C, wherein the binding partners C of every kind of receptor binding agent can be by multimerization
The binding site Z of reagent is (reversible) to be combined,
(ii) at least one polymerisation agent, at least one polymerisation agent, which includes, is used for every kind of receptor binding agent
The binding partners C at least one binding site Z, wherein the binding partners C of every kind of binding reagents with it is described more
The binding site Z of polymerizing agent can form reversible keying,
(iii) detectable label, the detectable label be bound to or can be bound to receptor binding agent (i) and/or
Polymerisation agent (ii).
Brief description
Non-limiting example and accompanying drawing are taken into consideration and will be better understood from the present invention with reference to detailed description of the invention, its
In：
Fig. 1 is shown with Fab fragments and (also existed as (at least one) receptor binding agent for Fab fragments and generally
In one embodiment of the present of invention, receptor binding agent contain for acceptor molecule single binding site) reversible dyeing
The principle of method.Fig. 1 a show that Fab fragments are used as the illustrated examples of receptor binding agent, and wherein Streptavidin binding peptide isFor the binding partners C of receptor binding agent.In this embodiment, Streptavidin binding peptide is merged or sewed
Fab fragments are closed so as to form receptor binding agent.In example shown in Fig. 1 a, polymerisation agent is Streptavidin mutation egg
In vain (), it includes at least two binding site Z at least one binding partners C.Scheming
In example shown in 1a, polymerisation agent carries fluorescence labeling such as phycoerythrin.In Fig. 1 b example, together illustrate again
Fab fragments and the polymerisation agent being conjugated on the magnetic bead as mark.Using Streptavidin mutain and Fab fragments it
Between via Streptavidin binding peptide multivalence combination compound formed reversible decoration method be also referred to as " Fab- polies herein
Body dyes ".Fig. 1 c show the schematic overview of this reversible Fab- polymers decoration method.In this example, from being present in blood
Dyeing/separation target cell in cell mixture in liquid sample.For being bound to the cell receptor being present on target cell, Koff
Speed is about 0.5 × 10-4sec-1Or bigger Fab fragments pass through Compound is formed can
Inverse multimerization.Target cell by multivalence combination compound (by make as shown in Fig. 1 step cs a) cell and polymerisation agent and by
Body binding reagents contact) dye and alternatively separated with lacking other cells of homology cell surface receptor molecule, for example, sharp
With fluorescence activated cell sorts art, (mark is " FACS in Fig. 1 step cs b)TM”).With withPeptide competitionThe target cell of part Bio subsequent treatment separation dyeing causes Fab fragments to be moved up from polymerisation agent
Position, thus cause polymerisation agent (Compound) the release (such as Fig. 1 step cs c) shown in) from target cell.
The Fab fragments of reservation, withIt is not compound, due to its high KoffSpeed will solve in rational time window (spontaneously)
From, and can be removed (shown in such as Fig. 1 step cs d)) from target cells completely by washing.Washing can be such a
Carried out under volume, Fab fragments are diluted at least equal to Fab fragments and homologous cell by the volume during each washing step
Compatibility dissociation constant (the K of combination between surface receptor moleculed) concentration.Fig. 1 d show similar to that of Fig. 1 c dyed
Journey, wherein polymerisation agent are conjugated on magnetic bead and wherein dyeing/separating step is included via thereon with reference to staining cell
Magnetic pole will separate in staining cell never staining cell.
Fig. 2 shows the binding characteristic needed for the reversible dyeing of Fab polymers.Fig. 2 shows to be contaminated with different anti-CD4Fab mutant
The facs analysis of the anti-CD4 Fab polymers of color, the anti-CD4Fab mutant of difference is for Fab fragments with dividing as acceptor
The CD4 of son combination has increased Koff- speed (summary of the binding kineticses of Fab fragments is listed in table 1).Peripheral blood mononuclear
Cell (PBMC) is tactic with being fused to two(commercial goods are entitled " One-STrEP-tag " for sequence；Knot
Close gametophyte C) anti-CD4Fab fragments (receptor binding agent) dyeing and with phycoerythrin mark
(Strep-Tactin PE；IBA GmbH,Germany；Include at least two binding site for binding partners C
Z polymerisation agent) multimerization, then before being handled with Bio (secondary series) or (the 3rd row) is analyzed afterwards.
Then after only using Strep-Tactin PE (uncombined Fab fragments) (the 4th row) to remove the washing step of Fab- monomers, detection
The Fab- monomers (the Fab fragments i.e. as receptor binding agent) retained on target cells.After the reversible dyeing of above-mentioned cell
The dyeing (the 5th row) of Fab polymers is carried out again, and to control the completion that biotin removes, otherwise the biotin will hinder such as
Reservation Fab- monomers shown in 4th row only use the dyeing of Strep-Tactin PE (uncombined Fab fragments), so as to cause mistake
Experiment conclusion.Or cell monomer Fab fragments culture, washing and with Strep-Tactin(first, i.e., it is most left
The row on side) dyeing after analyzed.Show CD3+ T cells living.Numeral in point diagram represents the percentage of door inner cell.
Fig. 3 shows the alternative of Fig. 2 facs analysis, i.e. Fab polymers dye reversible western blot analysis.
CD4 Fab polymers using the different anti-CD4 Fab- mutant for being listed in table 1 and further illustrating such as Fig. 2 withPE(IBA GmbH,Germany) generation.PBMC is incubated together with different CD4 Fab polymers
Educate, and the consistent cell dyeing in each sample (is contrasted, solid line, which represents, not to be contaminated with flow cytomery with light histogram
Cytochrome).Then, cell is handled with Bio and then washed.Finally, scrubbed cell cracking, and use high specific
It is anti-Antibody (StrepMAB-Classic Horse Radish Peroxidase (HRP) conjugate；IBA
GmbH,Germany) analyze the reservation Fab- monomers for dissolving (S) and undissolved (I) fraction.It is consistent amount of in order to control
The application of insoluble cellular material, while with first antibody (the Santa Cruz for beta-actin from rabbit
Biotechnology Inc., Santa Cruz, USA) and it is conjugated to the HRP secondary antibody for rabbit Ig (immunoglobulin)
(Sigma, St.Louis, USA0 detect beta-actin.
Detailed description of the invention
The present invention provides the side of a kind of target cell for making to have acceptor molecule in cell surface or the reversible dyeing of target cell group
Method.In comparison with the method for being described in United States Patent (USP) 7,776,562 or international patent application WO 02/054065, the present invention is to be based on
Such discovery, in the method, receptor binding agent are combined not with the low compatibility of the acceptor molecule on target cells
It is that invertibity is necessary.On the contrary, present invention has discovered that not considering bond strength, it is meant that no matter receptor binding agent and acceptor
Dissociation constant (the K combined between moleculed) whether there is low compatibility, such as in KdIt is about 10-3To 10-7In the range of M, or
It is no that there is high-affinity, such as in KdIt is about 10-7To 10-10In the range of M, target cell can be by reversible dyeing, as long as acceptor knot
Reagent is closed via the dissociation of the combination of binding site B and acceptor molecule to occur enough to fast.With KoffSpeed (also referred to as acceptor knot
Close the dissociation rate constant of combination of the reagent (via binding site B) between acceptor molecule) represent, KoffSpeed is about 0.5 ×
10-4sec-1Or it is bigger, about 1 × 10-4sec-1Or it is bigger, about 2 × 10-4sec-1Or it is bigger, about 3 × 10-4sec-1Or it is bigger, about 4
×10-4sec-1Or it is bigger, about 5 × 10-4sec-1Or it is bigger, about 1 × 10-3sec-1Or it is bigger, about 1.5 × 10-3sec-1Or more
Greatly, about 2 × 10-3sec-1Or it is bigger, about 3 × 10-3sec-1Or it is bigger, about 4 × 10-3sec-1Or it is bigger, about 5 × 10-3sec-1Or
It is bigger, about 1 × 10-2sec-1Or it is bigger, or about 5 × 10-1sec-1It is or bigger.Herein in regard to KoffSpeed, konSpeed or KdWhen institute
Referred to term " about " comprising ± 0.1%, ± 0.2%, ± 0.3%, ± 0.4%, ± 0.5%, ± 0.7%, ± 0.9%, ±
1.0%th, ± 1.2%, ± 1.4%, ± 1.6%, ± 1.8%, ± 2.0%, ± 2.2%, ± 2.4%, ± 2.6%, ±
2.8%th, ± 3.0%, ± 3.5%, ± 4.0.%, ± 4.5%, ± 5.0%, ± 6.0%, ± 7.0%, ± 8.0%, ±
9.0%th, ± 10.0%, ± 15.0% or ± 20.0% error margin.
Herein, it should be noted that between receptor binding agent L and its acceptor P (such as cell surface receptor molecule)
The formation of compound (C) can be described as two famous state procedures
Corresponding dissociation constant KdIt is defined as
Wherein [P], [L] and [C] is acceptor, receptor binding agent (part) and corresponding at a given temperature and pressure
Compound balance molar concentration.Dissociation constant KdAlso the speed i.e. positive reaction speed of compound combination/formation can be expressed as
(on-rate) constant (kon) (also referred to as association rate constant) and complex dissociation be back reaction speed (off-rate) constant
(koff) (also referred to as dissociation rate constant) ratio
Kd=koff/kon
In an application of the invention, thermodynamics and kineticses constant Kd、Ka、konAnd koffValue refer to its " standard conditions "
Under measured value, " standard conditions " be temperature be 25 DEG C, atmospheric pressure 1.013bar.
In the present invention, it is as noted above, it has been found that receptor binding agent is specifically bound with acceptor molecule via it
The k of site B combinationoffSpeed [s-1] determine by the further corresponding receptor binding agent containing binding partners C
The invertibity of target cell dyeing, wherein binding partners C can be combined by polymerisation agent defined herein.koffScope can
With based on for example, the to be dyed and target cell being optionally purified and corresponding experiment condition are selected in the range of.Examine
Consider the half-life period T of the compound (C) between receptor binding agent L and acceptor molecule P1/2Ln2/k can be expressed asoff=0.693/
koffAnd koffFor 0.5 × 10-4sec-1, the concentration reduction half consumption of the compound between receptor binding agent and acceptor molecule
13860 seconds or 231 minutes or 3.85 hours, wherein it is assumed that dilution is sufficiently so as to the receptor binding agent and acceptor point of dissociation
Recombining for son can be ignored.In order to realize the identical reduction of complex concentration, work as koffFor 1.0 × 10-4sec-1When
Consume 6390 seconds (or only 106.5 minutes or 1.775 hours), koffFor 2.0 × 10-4sec-1When consumption 3465 seconds (or 57 minutes,
It is less than 1 hour), koffFor 4.0 × 10-4sec-1When consume 1732 seconds (or about 28 minutes).Therefore, for thin in dyeing target
Born of the same parents' other cells from sample separate multivalent complex that is rear and being formed between polymerisation agent and receptor binding agent
Rupture after the washing step that carries out, can be based on wanting staining cell to combine examination to the sensitive Sexual behavior mode cell receptor of external action
Agent.For example, for quite sensitive cell, can use has at a relatively high koffSpeed, such as larger than 4.0 × 10-4sec-1's
Receptor binding agent so that after the rupture of multivalence combination compound, most receptor binding agents can be removed in 1 hour
(as described above, the concentration of compound was down to the 25% of initial concentration in 56 minutes, wherein it is assumed that because fully dilution recombines
Effect can be ignored).For stronger cell, can use has relatively low koffSpeed, such as 1.0 × 10-4sec-1's
Receptor binding agent, correspondingly its dissociation need more times (in this case it is necessary to 212 minutes or about 3.5 hours
To dissociate and remove 75% acceptor molecule binding reagents).(for example, being matched somebody with somebody by that can upset to combine after polymerisation agent dissociation
The competitor of reversible keying between even body C and polymerisation agent), washing constant temperature and can also be stirred in certain buffer solution volume
Mix at least twice half-life period T1/2Receptor binding agent used is diluted to concentration as dissociation by lower progress, the buffer solution volume
Constant (Kd) at least 10 times below.Then the receptor binding agent of dissociation is removed, for example, being settled by target cell, and is discarded
Clear liquid.In an optional embodiment, it can repeat above-mentioned to washed once or twice or repeatedly remove receptor binding agent
So as to be removed close to complete.Herein, it should be noted that when referring to the target cell of dyeing, term " washing " used refers to
Make target cell (being accompanied by the rupture of multivalent combination compound or after the rupture of multivalent combination compound) and the foot of dyeing
The lavation buffer solution contact of amount, suitable a period of time is incubated with lavation buffer solution so that receptor binding agent is from acceptor molecule
Dissociate and then separated by appropriate program from lavation buffer solution (it thereby eliminates the receptor binding agent of dissociation), example
Such as the sedimentation of target cell and the removal of supernatant, or the filtering of such as lavation buffer solution.Washing will be generally to that will dye or divide
From cell viability or the buffer solution that not adversely affects of state in carry out, i.e. washing is entered under the conditions of physiology is acceptable
Row (such as physiology is subjected to buffer solution, suitable temperature, for example, inferior 4 DEG C, 15 DEG C or 25 DEG C (that is, room temperature)).Suitable for experiment
Wash conditions can be easily empirically determined by those of ordinary skill in the art.If for example, koffThere is one close to 5.0
×10-4sec-1Of a relatively high value, and dye cell it is insensitive to high temperature (relative), then washing can be at room temperature
Carry out, so that receptor binding agent faster dissociates from staining cell.Alternatively, if the cell of dyeing is to higher temperature
Relative sensitivity, then washing can be carried out at 4 DEG C so that the dissociation of receptor binding agent/removal consumption long period, but cell
Functional status or viability will not be affected.In certain embodiments, it is preferred that temperature, which is 4 DEG C or at least less than 15 DEG C,
So as to preventing any change of target cell physiological status.
Herein, it should be noted that receptor binding agent can both contain (any) of specific binding acceptor molecule
Single binding site B, receptor binding agent can also contain two or more this binding sites B, if acceptor molecule via
The k of (each) binding site B combinationoffTotal value be about 0.5 × 10-4sec-1It is or bigger.Therefore, receptor binding agent can be with
It is monovalent (for example, the artificial binding molecule (albuminous or other) of the antibody fragment of unit price or unit price, is such as transported based on lipid
Carry the mutain of the polypeptide of protein family (also referred to as), or the molecule such as antibody of divalence or wherein
The all retained fragment in two basic change site such as F (ab ')2Fragment.Receptor binding agent can also be pentamer IgM molecules, bar
Part is the k of whole IgM moleculesoffSpeed is 0.5 × 10-4sec-1It is or bigger.
The k of receptor binding agent and acceptor moleculeoff- speed and certain konSpeed can also be surveyed by the method for standard
It is fixed, such as surface plasma resonance (SPR), for example, using BIAcore technologies (SPR；The such as Jonsson, U. (1991)
Biotechniques,11,620–627).This assay method is in the range of those skilled in the art's general knowledge.Generally, this
One measure is carried out at 25 DEG C by surface plasmon resonance assay, and acceptor molecule is consolidated under appropriate receptor binding agent concentration
It is scheduled in corresponding sensor chip surface.Part (that is, receptor binding agent), for example, (being generally near with various concentrations from most
Just characterize the K of the estimation of measuredValue) the Fab fragments of chip are applied to, the flow velocity used is in the range of μ l/min.
As an illustrative example, CD4 (as acceptor molecule) is mentioned herein and is derived from monoclonal antibody 13B8.2
Fab fragments combine koffRate determination.United States Patent (USP) 7,482,000 or Bes, C. are such as described in, waits .J Biol Chem
In 278,14265-14273 (2003), koffSpeed can use the instrument (BIAcore of BIAcore 2000 with other kinetic parameters
AB, Uppsala, Sweden) determined by surface plasma resonance at 25 DEG C.The CD4 of restructuring can be used and said according to manufacturer
Bright book is covalently fixed on CM5 sensor chip surfaces using amine coupled method.Compare reference surface (control reference
Surface) can be prepared using the unimplanted CD4 of identical chemical treatment flowing pool surface.Then 10mM Hepes are being included
(pH7.4), 3mM EDTA, 150mm NaCl and 0.005% nonionic surfactant P20 (BIAcore AB) buffering are molten
Recombinant Fab mutant in liquid can be injected on flow cell with 5 to 20 μ g/ml concentration, and dissociates the stage molten with 5mM HCl
After the regeneration step of liquid.Flow rate can be set as 30 μ l/min.All sensing figures can by from control master meter
Subtraction signal corrects in face.Data can be fitted to 1 comprehensively using the softwares of BIAevaluation Version 3.2:
1Langmuir isothermal binding curves.
As another illustrative example, ks of the anti-CD30Fab for recombinant C D30A antigen fragments is mentioned hereinoffSpeed
The measure of rate can wait " Functional humanization of an anti-CD30 Fab by Schlapschy, M.
fragment for the immunotherapy of Hodgkin's lymphoma using an in vitro
Evolution approach " Protein Eng Des Sel 17,847-860 (2004) surface plasma body resonant vibration is surveyed
Amount.The measure is carried out in BIAcore system X (BIAcore, Uppsala, Sweden).Buffer solution is handed over by gel filtration
After being changed to pH3.85 10mM sodium acetates, the CD30A of purifying is diluted to 50 μ g/ml and using amine coupling reagent box (BIAcore)
It is fixed on the CM5 sensor chips of " research grade ", produces 1700 fixed response units (RU).With a series of suitable concns
The recombinant Fab fragment of purifying is applied to PBS/P and (includes 0.005% surfactant P-20 PBS；BIAcore).Even
The lower formation for observing compound of buffer stream effect of 5 continuous μ l/min.The phase that sensing figure is measured by subtracting control nil trace
Induction signal is corrected, and the control blank uses to also online.Chip is 25 μ l/min 1mM NaOH's by applying flow velocity
2ml pulses, then balance and regenerate by using PBS/P.Kinetic parameter can use BIAevalution softwares V 3.0 again
(BIAcore) the 1 of baseline drift is passed through:1 (Langmuir) binding model determines.
Method of the present invention is returned, as noted above, parent of the receptor binding agent to cell surface receptor molecule
Low compatibility is needed not be with property.(see also experimental section) on the contrary, between the receptor binding agent and the acceptor molecule
With reference to dissociation constant (kd) may range from about 10-2M to about 10-8M, or about 10-2M to about 10-9M, or about 10-2M to about 0.8
×10-9M, or about 10-2M to about 0.6 × 10-9M, or about 10-2M to about 0.4 × 10-9M, or about 10-2M to about 0.3 × 10-9M, or
About 10-2M to about 0.2 × 10-9, or about 10-2M to about 0.15 × 10-9M, or about 10-2M to about 10-10As long as combined in multivalence multiple
After the rupture of compound, in a time window and under the acceptable wash conditions of cell to that will dye or separate, koffPermit
Perhaps the removal of receptor binding agent.In one embodiment, the combination between the receptor binding agent and the acceptor molecule
Dissociation constant (Kd) may range from about 10-7M to about 10-10M, or about 10-7M to about 0.8 × 10-9M, or about 10-7M is to about
0.6×10-9M, or about 10-7M to about 0.3 × 10-9M, or about 1.1 × 10-7M to about 10-10M, or about 1.1 × 10-7M is to about
0.15×10-9M, or about 1.1 × 10-7M to about 0.3 × 10-9M, or about 1.1 × 10-7M to about 0.6 × 10-9M, or about 1.1 ×
10-7M to about 0.8 × 10-9M.Herein it should be noted that for immunoglobulin class such as antibody or Fab- fragments and its phase
The combination for the antigen answered, it is known that kon- speed is up to about 5 × 106M-1s-1(see for example, Lee etc., Mol.Biol. (2004)
340,1073-1093, wherein Fig. 4 shows Fab fragments G6-23 kon- speed is 5.6 × 106M-1s-1Or, for example, Kwong etc.
J.Mol.Biol.2008December 31；384(5):Reported in 1143-1156 or international patent application WO 2010/017103
The k of the Fab fragments of Anti-Human's NKG2D monoclonal antibodies and the IgG1 (see Kwong etc., above, table 1) as antibodyonIt is worth for 1
×105M-1s-1To 1.4 × 106M-1s-1).Thus, it is supposed that receptor binding agent such as Fab fragments have and the combination of acceptor molecule
konIt is worth for 5 × 106M-1s-1And KdFor 1x10-10, koffFor 5x10-4M so that this antibody molecule (or in general acceptor combines examination
Agent) it will be very suitable for (working as k for reversible decoration method of the present inventionoffFor 5x10-4sec-1When receptor binding agent and acceptor
The concentration of compound reduces half and only consumes 1386 seconds or 23 minutes between molecule, although compatibility KdFor 1x10-10M).Equally
Ground, as further example, if the k of the combination of receptor binding agent and acceptor moleculeonIt is worth for 5 × 105M-1s-1And Kd
For 1x10-9, koffAlso it is 5x10-4M, as a result also result in after the rupture of multivalence combination compound, receptor binding agent is thin from target
Quickish dissociation on born of the same parents.In this case, it can be with for example, by using Evolve-ment law (evolutionary
Method) such as phage display, receptor binding agent such as antibody fragment or such as lipocalin mutein egg are screened intentionally
(also compare Lee etc., this respect above) in vain.Therefore, if it is desirable to the receptor binding agent with ideal kinetics property can
To be obtained by this Evolve-ment law from the starting molecule of known amino acid sequence.Can also use method described herein in by
Body molecule has the receptor binding agent of low compatibility, so as to which the dissociation of the combination between receptor binding agent and acceptor molecule is normal
Number (Kd) value scope be 10-2To 10-7M。
The decoration method further comprises the non-target cell separation staining cell present in sample/cell mixture.Institute
Stating separation can be carried out as described in United States Patent (USP) 7,776,562 or international patent application WO 02/054065, and can be entered
One step include by rupture the combination between the binding partners C of receptor binding agent and the binding site Z of polymerisation agent from
The cell removes the dyeing.This bonding should be reversible, i.e. this is bonded in the bar for being adapted for carrying out claimed method
It can be broken under part.Dissociation constant (the K of combination between the binding partners C and the binding site Zd) can be with example
Such as, for about 10-2M to about 10-13In the range of M.Therefore, this reversible bonding can be with for example, the K havingdIt is about 10-2M
To about 10-13M, or about 10-5M to about 10-13M, or about 10-6M to about 10-10M, or about 10-3M to about 10-12M, or about 10-4M is to about
10-11M, or about 10-5M to about 10-10M.The K of this bondingdValue can also be determined by any appropriate method, for example, passing through
Fluorescence titration, equilibrium dialysis or the K being bonded formed as described above between receptor binding agent and acceptor moleculeoffRate determination
With reference to the surface plasma resonance of explanation.The polymerisation agent of dyeing result in the acceptor knot of dissociation from dissociation/removal of cell
Close reagent and therefore the whole multivalence combination compound comprising detectable label removes from prestained cell.In order to promote
The removal of receptor binding agent, using the corresponding lavation buffer solution of certain volume so that the concentration of the receptor binding agent of addition
Substantially less than its K answered with homoreceptor molecule phase interactiond.Or washing can be repeated several times with less volume, and gently stir
Mixing can be advantageous to prevent to recombine immediately.In one embodiment, acceptor is reduced with the lavation buffer solution of certain volume to combine
Reagent concentration is less than KdOr even more preferably still concentration is less than Kd1/10 so that more than 90% receptor binding agent is to dissociate
Form (being not associated with to acceptor molecule) exist or even more preferably still concentration is less than Kd1/100 so that more than 99%
Receptor binding agent exists in the form of dissociating or even more preferably still concentration is less than Kd1/1000KdSo that more than 99.9%
Receptor binding agent exist in the form of dissociating.
In some embodiments of the invention, receptor binding agent is selected so that it includes at least one binding partners
C, and polymerisation agent includes the knot of at least two binding site Z, at least three or at least four for the binding partners C
Close site Z.In an alternate embodiment of the invention, the receptor binding agent of two differences (species) can be used.The two acceptors combine
(for latter event, two receptor binding agents identifications are simultaneously by identical or different binding site B for each in reagent
So as to combine the different epitopes of same receptor molecule) combine identical acceptor molecule.It is in addition, every in two receptor binding agents
One has at least one binding partners C, and polymerisation agent has at least one binding site Z, for two differences
The binding partners C of each in receptor binding agent.It is, for example, possible to use two different receptor binding agents, it is all
Via identical binding site B combination identical acceptor molecules, but it has different binding partners C1 and C2.For example,
One receptor binding agent can have a binding partners C1 such as six-histidine-tagged, and another receptor binding agent has and tied
Close gametophyte C2 such as FLAG labels.In this case, polymerisation agent can include the only one knot for binding partners C1
Close site (Z1) and the only one binding site for binding partners C2.Due to two knots in polymerisation agent altogether be present
Site is closed, compatibility effect remains to reach.This polymerisation agent can be, for example, biocompatible polymer is (for example, right
Revolve glucosides or other polysaccharide), a Fab fragment of the Fab fragments of anti-FLAG antibody and anti-hexahistidine tag antibody is conjugated
To the biocompatible polymer (being discussed in detail for polymerisation agent sees below).Regardless of whether containing only one or two or more
Multiple binding partners C one or two receptor binding agent is used for times of the present invention, binding partners C and polymerisation agent
Meaning is combined and can be all selected, as long as the binding site Z of binding partners C and polymerisation agent can be reversible in (multivalence) compound
Ground multimerization is so as to trigger compatibility effect, such as institute in United States Patent (USP) 7,776,562 or international patent application WO 02/054065
State.
In some illustrative property embodiments, gametophyte may be selected from following：
(a) binding partners C includes biotin, and polymerisation agent includes Reversible binding to the Streptavidin of biotin
Analog or avidin analog.
(b) binding partners C includes Reversible binding to Streptavidin or the biotin analog of avidin, and
Polymerisation agent includes Reversible binding to the Streptavidin, avidin, Streptavidin of the biotin analog
Analog or avidin analog, or
(c) binding partners C includes Streptavidin or avidin binding peptide, and polymerisation agent is comprising reversible
It is bound to Streptavidin, avidin, the Streptavidin of Streptavidin or the avidin binding peptide
Analog or avidin analog.In some of these embodiments, binding partners can include strepto-
Avidin binding peptide Trp-Ser-His-Pro-Gln-Phe-Glu-Lys, and the polymerisation agent includes Streptavidin class
Like thing Va144-Thr45-Ala46-Arg47Or Streptavidin analog Ile44-Gly45-Ala46-Arg47, both multimerizations
Reagent is for example described in United States Patent (USP) 6, and 103,493, commercially available trade mark isStreptavidin binding peptide can be with
It is, for example, single peptide, is such as described in United States Patent (USP) 5,506,121 , for example, or with such as international monopoly
Apply for the Streptavidin binding peptide of two or more tactic single binding molecules described in WO 02/077018.
In further embodiments, a binding partners C, wherein binding partners C and the multimerization be only used
Being incorporated in the presence of bivalent cation between at least two binding site Z of reagent occurs.At one of this embodiment
In illustrative example, binding partners C includes Calmodulin-binding peptide, and polymerisation agent includes such as such as United States Patent (USP) 5,
Polymer calmodulin described in 985,658.Or binding partners C can include FLAG peptides, and the polymerisation agent can
Comprising the antibody for being bound to FLAG peptides, such as FLAG, it is bound to such as United States Patent (USP) 4, the monoclonal antibody described in 851,341
4E11.In another illustrative example, binding partners include oligo-histidine label, and polymerisation agent is included and is bound to
The antibody or transition metal ions of oligo-histidine label.The rupture of all these combination compounds can pass through metal ion-chelant
To complete, as calcium chelate, such as by adding EDTA or EGTA.Calmodulin, antibody such as 4E11 or chelating metal ion or trip
From chelating agent can by conventional method by multimerization, such as by biotinylation and with Streptavidin or avidin or
Its polymer or by the way that carboxylic acid residues are imported in polysaccharide, the polysaccharide such as glucan, substantially such as Noguchi, A.,
Takahashi,T.,Yamaguchi,T.,Kitamura,K.,Takakura,Y.,Hashida,M.&Sezaki,H.(1992).
“Preparation and properties of the immunoconjugate composed of anti-human
colon cancer monoclonal antibody and mitomycin C dextran
The first step is described in conjugate.Bioconjugate Chemistry 3,132-137 " and uses routine in second step
Carbodiimide chemist make calmodulin or antibody or chelated metal ions or free chelating agent via primary amine groups and polysaccharide such as Portugal
Carboxyl coupling on glycan main chain.
In such an embodiment, the binding partners C and the polymerisation agent at least two binding sites Z
Between combination can be ruptured by metal ion-chelant.Metal ion-chelant can be completed for example, by adding EDTA.
In the method for the invention, polymerisation agent can also be Streptavidin or avidin or strepto- parent
With element or the oligomer or polymer of any analog of avidin.The oligomer or polymer can be handed over by polysaccharide
Connection.In one embodiment, Streptavidin or avidin or Streptavidin or avidin is similar
The oligomer or polymer of thing by residual carboxylic acid group being imported in polysaccharide such as glucan to prepare, substantially as " Noguchi, A.,
Takahashi,T.,Yamaguchi,T.,Kitamura,K.,Takakura,Y.,Hashida,M.&Sezaki,H.(1992).
“Preparation and properties of the immunoconjugate composed of anti-human
colon cancer monoclonal antibody and mitomycin C dextran
In conjugate.Bioconjugate Chemistry 3,132-137 " described by the first step.Then use in second step often
The carbodiimide chemist of rule makes Streptavidin or antibiotin or its analog via internal lysine residue and/or free N-
The primary amine groups of end are coupled on the carboxyl of dextran backbone.But Streptavidin or avidin or strepto- parent
Oligomer or polymer with the crosslinking of element or any analog of avidin also can be by connecting via difunctional
Thing such as glutaraldehyde (glutardialdehyde) is crosslinked acquisition by the other method described in document.
In another embodiment of decoration method of the present invention, binding partners C includes antigen, and polymerisation agent bag
Containing the antibody or antibody fragment for the antigen.This antigen can be, for example, epitope tag.Suitable epitope tag
Example includes, but not limited to Myc- label (sequences：EQKLISEEDL), HA- labels (sequence：YPYDVPDYA), VSV-G- is marked
Sign (sequence：YTDIEMNRLGK), HSV- labels (sequence：QPELAPEDPED), V5- labels (sequence：GKPIPNPLLGLDST)
Or glutathione-S-transferase (GST), only enumerate several.In another embodiment, binding partners C includes maltose
Associated proteins (MBP), chitin-binding protein (CBP) or thioredoxin are as antigen.In these cases, polymerisation agent
The compound formed between at least two binding sites Z and antigen of (antibody) can be by adding free antigen i.e. such as Myc-
The free peptide or floating preteins (such as MBP or CBP) of label or HA- labels (epitope tag) destroys.Herein, it is necessary to pay attention to
Be in FLAG- label (sequences：DYKDDDDK) it is used as binding partners and polymerisation agent to include with reference to the anti-of FLAG labels
In the case of body or antibody fragment, also this reversible keying can be destroyed by adding free FLAG peptides.
In the method for the invention, be specifically bound to the acceptor molecule receptor binding agent it is described at least
One binding site B can be such as antibody or bivalent antibody fragments such as (Fab)2'-fragment, bivalent single-chain Fv fragments.It also may be used
To be the artificial binding molecule of divalent protein, be such as otherwise known as the dimer lipocalin mutein egg of " duocalin "
In vain.In other embodiments, receptor binding agent can have single binding site B, be unit price.Monovalent receptor binding reagents
Example include, but not limited to monovalent antibody fragments, there is the proteinacious binding molecule or MHC point of antibody sample binding characteristic
Son.
The example of monovalent antibody fragments includes, but are not limited to Fab fragments, Fv fragments and Single-Chain Fv Fragment of Murine (scFv).
Specifically bind the proteinacious with antibody sample binding characteristic that can be used as receptor binding agent of acceptor molecule
The example of binding molecule includes, but not limited to aptamers, the mutain of polypeptide based on lipocalin protein family, restructuring
Albumen (glubody), the albumen based on ankyrin, albumen, adnectin, Avimers based on crystallization support
(avimer), EGF- spline structures domain, kringle domain (Kringle-domain), the type domain of fibronectin I, fibronectin II
Type domain, the type domain of fibronectin III, PAN structure domain, G1a domains, SRCR domains, Ku Nici (Kunitz)/ox
Trypsin inhibitor domain, Tendamistat (tendamistat), Kazal- type serpin structures
Domain, three leaves (P-type) domain, vWF ELISA c-type domain, anaphylatoxin spline structure domain, CUB domains, first shape
The type repetitive sequence of gland globulin I, ldl receptor A class formations domain, Sushi domains, Link domains, the type of thrombospondin I
Domain, immunoglobulin domains or immunoglobulin like domain (for example, domain antibodies or camel heavy chain antibody), C
Type Lectin domain, MAM domains, von Willebrand disease A types domain, growth regulator B domain, the sulphur of WAP types four or two
Compound Core domain, F5/8C types domain, hemopexin domain, SH2 domains, SH3 domains, layer adhesion
Protein type EGF spline structures domain, C2 domains, " Kappabodies " (" the Design and construction of such as Ill. a
hybrid immunoglobulin domain with properties of both heavy and light chain
variable regions"Protein Eng 10:949-57 (1997)), " the miniantibody " (such as Martin " The
affinity-selection of a minibody polypeptide inhibitor of human interleukin-
6"EMBO J 13:5303-9 (1994)), " Janusins " (" the Bispecific single such as Traunecker chain
molecules(Janusins)target cytotoxic lymphocytes on HIV infected cells"EMBO J
10:" the Janusin such as 3655-3659 (1991) and Traunecker:new molecular design for bispecific
reagents"Int J Cancer Suppl 7:51-52 (1992)), nano antibody, adnectin, tetranectin, microbody
(microbody), affilin, affine body or ankyrin, crystallin, knottin, ubiquitin, zinc finger protein, autofluorescence
Albumen, ankyrin or ankyrin repeat protein or rich in leucine repetitive proteins, avimer (Silverman, Lu Q, Bakker
A,To W,Duguay A,Alba BM,Smith R,Rivas A,Li P,Le H,Whitehorn E,Moore KW,
Swimmer C,Perlroth V,Vogt M,Kolkman J,Stemmer WP 2005,Nat Biotech,Dec；23(12):
1556-61,E-Publication in Nat Biotech.2005Nov 20edition)；And pass through people's acceptor domain
The multivalence avimer albumen that the extron shuttle evolution of family comes, as be also depicted in Silverman J, Lu Q, Bakker A, To
W,Duguay A,Alba BM,Smith R,Rivas A,Li P,Le H,Whitehorn E,Moore KW,Swimmer C,
Perlroth V,Vogt M,Kolkman J,Stemmer WP,Nat Biotech,Dec；23(12):1556-61,E-
Publication in Nat.Biotechnology.2005Nov 20 editions.
In the method for the invention, the mixture comprising target cell is set to be contacted with receptor binding agent and polymerisation agent
It can be carried out at any suitable temperature, such as in United States Patent (USP) 7,776,562 or International Application WO 02/054065
Description.Under normal circumstances, the mixture comprising target cell is made to be contacted with receptor binding agent and polymerisation agent and described afterwards
The removal of reagent can be carried out at such temperatures, at such a temperature, in the case where T cell will be colored or separate, substantially
On be not likely to result in the target cell such as activation of T cell phenotypic alternation and/or signal event and occur.In some preferred implementations
In example, contact and carried out at temperature≤15 DEG C or≤4 DEG C.
In invention methods described, almost it is any it is described with it is at least one be used to dyeing or separate target cell it is common by
The target cell of body molecule all can be used.In order to reach compatibility effect, acceptor molecule generally on target cells with two or
More copies are present.In an exemplary embodiment, target cell is mammalian cell or eucaryon or prokaryotic.Equally, it is fixed
At least one identical (specificity) acceptor of adopted target cell group can combine examination for the acceptor that can be targeted as described above
Any acceptor of agent.For example, acceptor can be to define cell mass or the antigen of subgroup, such as haemocyte group or subgroup, such as lymphocyte
(such as T cell, t helper cell, for example, CD4+T auxiliary cells, B cell or NK), monocyte or stem cell,
Such as CD34 positive peripherals hemocytoblast or Nanog or Oct-4 expression stem cell.The example of T cell is included such as CMV specificity
CD8+ T lymphocytes, cytotoxic T cell, memory T cell and regulatory T cells (Treg).A Treg illustrative reality
Example is CD4+CD25+CD45RA Treg cells, and an illustrative example of memory T cell is CD62L+CD8+Specific center
Memory T cell.Acceptor may also be the label of tumour cell.Herein, it should be noted that " target is thin for terms used herein
Born of the same parents " include all biological entities/vesicas, wherein film (double-layer of lipoid can also be made) by it is internal separated with external environment condition and comprising
Specific acceptor molecule on biological entities surface.The example of this entity includes, but not limited to virus, liposome and thin
Born of the same parents' device such as mitochondria, chloroplaset, nucleus or lysosome.
Once being separated, then target cell group is characterised by that receptor binding agent substantially completely moves from the acceptor
Except (usually less than test limit, such as detected with FACS).The complete removal of receptor binding agent is by using as above institute
State with high koffWhat the reversible multimerization receptor binding agent of speed was completed.By doing so it is possible, the not side of being purified can be provided
The individual target cell group with functional status that method changes (by being defined with common specific receptor).
Inside is separated and is further characterised as with external environment condition by target cell group or wherein film (can also be double-layer of lipoid)
In any other biological entity group that surface includes common specific acceptor molecule any purifying for using can be being removed afterwards
Reagent (receptor binding agent；Polymerisation agent；Label) in the case of the fact that purified by the method for the invention carry
Following supervision advantages (if being cell or organelle beyond target, the advantage that its physiological status will not be changed) are supplied, i.e.,
When this purified biological entities are as medicine, purified reagent will not be administered into patient.In this case, regulatory authority
If FDA (U.S.) or EMEA (Europe) in the case that purified reagent is administered together with the medicine for cell or liposome than needing
Relatively low cost is needed to limit with regard to the production process of the purified reagent.Therefore, clear and definite technical advantage exists in the present invention
The method of the entity purifying, the physiological status of the entity is handled like that without the image of Buddha for such as liposome, if this lipid
Body must purify and as if medicine.
Mark for staining cell detection can be for diagnosing and any mark in analysis method.Preferable mark
The property of cell to dye or separate has no adverse effect.The example of mark has fluorescence labeling (for example, phycoerythrin, algae
Azurin, cumarin or rhodamine, only enumerate several), it is magnetic mark, coloured label (chromophoric label), suitable
Spin label (spin label) or radioactive label for electron spin resonance/electron paramagnetic resonance (EPR).Mark can
To be bound to receptor binding agent and/or polymerisation agent.Mark can directly be marked, that is, be bound to multivalence as specified above
With reference to one in compound member.In this case, the mark can be with, for example, covalent coupling (conjugated) to or by
Body binding reagents or polymerisation agent (with reference to the polymerisation agent for carrying fluorescence labeling phycoerythrin as shown in Figure 1).Can
Selection of land, mark can be indirect labellings, i.e., mark is combined with other reagents, and the reagent can be bound to as specified above more in turn
One in valency combination compound member.This mark can multivalence combination compound formation before, among or add afterwards.This
The example of kind of indirect labels is two, the three or four-NTA for including fluorescent dye, such as Lata .Am.Chem.Soc.2005,
Described by 127,10205-10215 or Huang etc., Bioconjugate Chem.2006,17,1592-1600.The mark energy
Non-covalent binding (via metal-chelating) is to oligo-histidine label.Therefore, in this example, receptor binding agent and/or
Polymerisation agent can carry oligo-histidine label, and (for example, the Fab fragments as receptor binding agent, it has oligomerization group
His tag, such as it is fused to hexahistidine tag or such as conduct of Streptavidin mutain of CH1 or CL- domain Cs-end
Polymerisation agentIt has N- the or C- ends for being fused to one available subunit), thus it is used for
Non-covalent binding Lata etc. is above or the above-described fluorescent dye compound based on NTA such as Huang.This Non-covalent binding
Mark is not necessarily required to be bound to its target (receptor binding agent and/or multimerization examination before multivalence combination compound is formed
Agent), but also can when multivalence combination compound is formed or multivalence combination compound formed after be added to sample in.This
Kind Non-covalent binding mark can also add after multivalence combination compound is bound to target cell.Except the fluorescent dye comprising NTA:
Oligo-histidine combination pair described above, or other any specific bindings for example, carrying to such as, can be used for indirect labelling
The digoxin of fluorescence or other marks (digoxigenin) and anti digoxin antibody or antibody fragment.In this case, acceptor
Binding reagents and/or polymerisation agent be conjugated to digoxin or carry it is selected be bound to (via digoxin) polymerisation agent or
The anti digoxin antibody or antibody fragment of the mark of receptor binding agent/combined with aforementioned substances.
In the embodiment of the method for the invention, target cell be colored and optionally by least two it is different (in advance
Selection) group of acceptor molecule is isolated.The separation for representing the target cell more than a coreceptor can be by with order
Mode implements the method for the invention progress, wherein using corresponding receptor binding agent in each circulation.In this case,
Identical mark can be used for each circulation.Or when isolabeling is not used for each acceptor molecule, mark is while target cell
It is possible.Such sequential cell enrichment and/or the dyeing of (simultaneously) multi-parameter use two kinds of different receptor binding agents, every kind of
Receptor binding agent includes at least the one of the pre-selection acceptor molecule being specifically bound in the group of at least two different acceptor molecules
Individual binding site B.Illustratively say, at least two different acceptor molecules can be different CD labels, such as CD4+CD25+
Regulatory T cells or CD62L+CD8+The central memory T cell of specificity.Cell can be, for example, T cells with antigenic specificity is such as
CMV- specific Cs D8+T cell, it can be by selecting CD8 first+T cell, then by second step dye and separate CMV- it is special
Property target group such as HLA-B8/IE1K199- 207- specific Cs D8+T cell and be separated.
In such an embodiment, second or any other acceptor molecules of the group of at least two different acceptor molecules are bound to
Receptor binding agent can further include at least one binding partners C.
Dyeing for Co receptor molecule, via the receptor binding agent and described the of binding site B specific bindings
Dissociation rate constant (the k of combination between two or any other acceptor moleculesoff) also have 0.5 × 10-4sec-1It is or bigger
koffSpeed.Alternatively, the method in United States Patent (USP) 7,776,562 or international patent application WO 02/054065, described
The receptor binding agent of two or any other acceptor molecules and described second or any other acceptor molecules of specific binding
Between combination dissociation constant (Kd) it may range from 10-2To 10-7M。
As discussed above, colouring method of the present invention can be used for being determined by least one common specific acceptor molecule be present
The separation of the cell mass of justice.The cell mass defined by least one common specific acceptor molecule be present can be, for example, T is thin
Born of the same parents group, including such as T cells with antigenic specificity group.The cell mass of these purifying can be used for such as adoptive transfer or immunotherapy.
The method of the invention can also be used for the measure of the functional status of T cell group, or T cell group purifying for body
Outer amplification.
The more detailed and disclosure according to United States Patent (USP) 7,776,562, the method for the invention allow target cell group such as T thin
The separation of the feature based on reversible dyeing course of born of the same parents group.The original function state of target cell such as T cell can identification and it is pure
Kept substantially after change.Therefore, the method for the invention has extensive benefit to basic research and clinical practice.
Preferable application example is as follows：
Basic research
The direct vitro study of functional status of the lymphocyte as included the T cell such as T cells with antigenic specificity group.It is proposed
The functional status of T cell group is high diversity and depends on condition inside specific in vitro, but suitable due to lacking
Detection instrument, these aspects are only somewhat to understand.For example, as described herein, can be to epitope specificity T using Fab polymers
Cell mass is identified and purified independently of its functional status.Surveyed however, the combination of irreversible reagent disturbs subsequent function
It is fixed.Reversible T cell dyeing such as use outfit Streptavidin binding peptide/The Fab fragments of reagent, are one
Kind allows the technology of the direct function vitro study of constant diversified T cell group.
The purifying of T cell group is for external efficient amplification.The sign of the T cell group obtained in vitro often needs further to exist
Amplification in vitro is cloned to T cell system or T cell.
Using Fab polymer technologies, the not isophenic subgroup in separable unicellular or different T cell groups, but institute
The combination for stating reagent and TCR disturbs amplification in vitro efficiency.This experiment problem is carried out by using reagent of the present invention can
Inverse T cell is dyed to solve, the reagent such as, Reagent.This Fab fragments
It can carry as having tactic two as binding partners described in International Patent Application Publication WO 02/077018
The Streptavidin binding peptide of individual or more single coupling unit.
The purifying of T cell group is for adoptive transfer experiment.Many experiment in vivo need to purify in immunological investigation
T cell adoptive transfer into receptor.The purity and generation of the T cell group of transfer may in the cell in separation process
Change all paid close attention in these experimental systems.The highest purity of cell mass is obtained by positive back-and-forth method, but is used for
The label (being usually antibody) of identification is difficult to remove and may interfere with inside then from the cell surface of separation to test knot
Fruit.
Using reagent of the present invention such asThe reversible T cell dyeing of reagent is permitted
Perhaps positive back-and-forth method and the removal of subsequent selection markers thing are applied in combination and can greatly improve adoptive transfer experiment.
Clinical practice
The purifying of T cell group is for efficient amplification in vitro.Generation (such as pathogen/tumour spy that human T-cell is or cloned
The opposite sex or autoreactive T cell) in many fields of clinical research, diagnosis and immunization therapy it is necessary.In vitro culture is normal
The problem for being commonly used for the condition standard of antigen-specific sexual stimulus is limited.Can be big to the improvement strategy of T cell group purifying
Big increase amplification in vitro efficiency, so as to allow to stimulate such as mitogen and anti-CD3 using antigen-independent.Using this
Invention, such as useOrReagent (as receptor binding agent) andPolymerisation agent, T cell group directly isolated ex vivo and can expand in vivo after reagent dissociation.This side
Method expection is more more efficient than using for example conventional irreversible MHC polymerisation agents, because the interfering bodies that the combination of reagent can be negative
The efficiency of outer T cell amplification.
T cell group for further functional analysis or controls from the purifying in amplification in vitro cell line or clone
Treat.The amplification in vitro of T cell needs to add antigen presenting cell or feeder cells into culture.For further function point
Analysis, especially for treatment use (such as adoptive transfer), it can help to the cell for removing these pollutions.Journey is selected for positive
Sequence (normally result is the purity of top), selection markers thing should remove from T cell surface, because it can interference function survey
Fixed or adoptive transfer.If T cell is used for vivo applications, selection markers thing is such as comprising can cause clinical complication such as allergy anti-
The material answered then must be removed further.As usedOrReagent/The reversible T cell dyeing of reagent meets all these standards.
For " direct adoptive immunotherapy " T cell group it is ex-vivo purified.Immediately will after the direct isolated ex vivo of T cell group
Cell, which is transferred in acceptor (without any further vitro propagation), has special clinical value.It is expected that be directly separated
Cell mass is more more effective than the cell of the culture for vivo purposes.The amplification in vitro T cell of high quantity is needed to carry out effectively
Adoptive transfer, this phenomenon most-likely due to T cell to caused by the adaptability of condition of in vitro culture.One of this process
Important clinical practice example is EBV and/or CMV- specific T-cells group in [otherwise] consumption T cell (T cell-
Depleted parallel purification in stem cell transplantation) and adoptive transfer, this, which is one, can be reduced substantially in transplant patient
The program of EBV and CMV- associated malignancies incidences.As usedOr
As receptor binding agent andThe dyeing of reversible T cell and separation as polymerisation agent are to be used for these
The most suitable method of clinical application.For example, the molecules of MHC I (being fused to Streptavidin binding peptide) permission is white from donor blood
The separation for the high-purity C MV specific C D8+T cells that cell obtains；Then this purifying cells, which are directly transferred to suffer from, jeopardizes life
(such as transplanted in the patient of the immunologic hypofunction of the CMV diseases of life through Allogeneic stem cell).Referring to Schmitt etc.,
(2011) content in TRANSFUSION, Volume 51, pp.591-599 on this respect.Other examples include being used for
After the Treg cell masses of the purifying of transfer, for treating autoimmune disease such as graft versus host disease(GVH disease) (GvHD) or graft
Repulsion or IDDM, enteritis or allergy.Treg target cells group for these purposes can be CD4+CD25+CD45RA+
Treg cells, these cells by using three receptor binding agents as specifically bind CD4 acceptor molecules, CD25 acceptor molecules or
The Fab fragments of CD45RA acceptor molecules are dyed/separated in a sequential manner.Other examples include natural killer cell via
CD56 acceptor molecules (also referred to as N-CAM (NCAM)) or candidate stem cell such as CD34+、CD59+、Thy1/
CD90+、CD38lo/-、CD133+Or Ckit/CD117+Candidate stem cell via corresponding cell surface receptor molecule separation.
Functional T cell diagnoses
MHC polymer technologies allow T cells with antigenic specificity directly ex vivo quantitative and Phenotypic characterization.But multimerization
Reagent and TCR combination cause (such as chronic viral infection [HIV, CMV, EBV, HBV, HCV], tomour specific in functional examination
Property T cell group) uses of purifying cells complicates.
As usedThe dyeing of reversible T cell and separation of reagent open
The gate of this T cell state is effectively evaluated in many clinical settings.
To further it be proved by the following experiment embodiment present invention.
Embodiment
Material and method
Blood sample
Fresh PBMC is obtained by centrifugation in Biocoll separating liquids from peripheral blood or buffy coat.Peripheral blood is
What health adult's donor from Medical Microbiology, immunology and hygiene research institute (Technical University at Munich) obtained, and
Buffy coat is residing for the anesthesiology research from Munich Germany Heart center (Bavaria state and Technical University at Munich)
Autologous donate blood what donor obtained.The informed consent form signed at donor, and the use of blood sample is according to state's laws
By local institutional review board (Ethikkommission der Medizinischender
Technischen) approval.
Generation (clone, expression, purifying) as the Fab fragments of receptor binding agent
Variable domains from monoclonal anti-CD 4 antibodies 13B8.2 use United States Patent (USP) 7,482,000 and Bes, C., etc.
Sequence described in J Biol Chem 278,14265-14273 (2003) is obtained by gene chemical synthesis.What is obtained afterwards is variable
Sequence is connected with the sequence of the coding heavy chain of human constant region Ch1 types 1 and kappa light chains, and heavy chain is arranged in carboxyl terminal and order
Two Streptavidin combination module (SAWSHPQFEK (GGGS) of row2GGSAWSHPQFEK) merge, the module conduct
One-STrEP- labels affinity tag from German IBA GmbH,It is commercially available.All clones by usingCloning system (IBA GmbH,Germany) and suitable for the Fab expression fusion vector carry out.
PCR mutagenesis is applied to introduce amino acid replacement.After clone, Fab-One-STrEP- tag fusion proteins are in coli strain
Expressed in JM83 (A.&Skerra,A.Expression of functional antibody Fv and Fab
fragments in Escherichia coli.Methods Enzymol 178,497-515(1989)).After periplasmic expression,
Fab fragments viaSuper fluidization tower (IBA) by One-STrEP- labels/Affinity chromatography
Method purifies (Schmidt, T.G.＆Skerra, A.Thesystem for one-step purification
and high-affinity detection or capturing of proteins.Nat Protoc 2,1528-1535
(2007)), discharged from the desthiobiotin for eluting corresponding Fab from post by dialysis, and be thus transferred to PBS
(- 20 DEG C) are stored in PBS pH7.4 in pH7.4 buffer solutions and at 4 DEG C or under frost.
Multimerization, dyeing and facs analysis
For dyeing of the CD4 acceptor molecule specificity target cell groups in cell mixture, using being marked with as fluorescence
The phycoerythrin of mark(IBA GmbH,Germany) and antibody 13B8.2 as described below
The monomer CD4 combination Fab-One-STrEP fragments of variant are used as polymerisation agent together.For facs analysis, make 5 × 106It is individual
PBMC and the Fab polymers as receptor binding agent and the 0.7 μ g as polymerisation agentPE(IBA
GmbH,Germany) it is incubated together using following proposal, the Fab polymers (include knot by 0.2 μ g Fab- fragments
Close site B) formed with the One-STrEP- labels (binding partners C) being furnished with.In addition, after FACS separation, each dyeing is thin
Multivalent complex in the part of born of the same parents is broken by adding biotin as described below.Control antibodies dyeing passes through corresponding antibodies
Carried out with application：Anti- CD4 (OKT4) (come from eBiosciences companies, San Diego, CA, the U.S.).The present invention's
After dyeing course, cell is then dyed with propidium iodide so as to identify cell living/dead.
5×106The experimental program of individual cell receptor binding agent and polymerisation agent dyeing
1. it is incubated 0.75 μ g Strep- with 0.2 μ g monomer CD4 combination Fab-One-STrEP fragments in the dark 4 DEG C
Tactin PE 30 minutes.
2.5×106The individual cell pre-cooled is with 10ml buffer solutions IS (in pH7.4 phosphate buffered saline (PBS) (PBS)
0.5%BSA (w/v), wherein PBS=0.86mM Na2HPO4 1.47mM KH2PO4, 137mM NaCl) and in 15ml reaction tubes
It is middle to wash to remove potential composition, such as example, the Bio of subsequent program can be disturbed.
3. cell is resuspended in 50 μ l buffer solutions IS and is transferred in the reaction vessel suitable for dyeing, such as 96 hole round bottoms
Microtiter plate.
4. the Fab-Streptamer preparations of the advance incubation obtained from step 1 are added in cell and by gently taking out
Suction is sufficiently mixed.
5. mixed liquor is cultivated 20 minutes at 4 DEG C in the dark.
6. cell is washed three times by centrifugal process (400xg, 2min) in 200 μ l buffer solutions IS.
7. present cell has been handled well and sorted for facs analysis or FACS, facs analysis is in CyAn ADP Lx
Carry out on (Beckman Coulter) and analyzed with FlowJo softwares (TreeStar).
The dissociation of multivalence nanocrystal composition and subsequent receptor binding agent Bio are from 5 × 106Removed in individual cell
Experimental program
1. collect 5 × 10 by centrifuging (400xg)6The cell (seeing above) of individual dyeing simultaneously is resuspended in including 1mM D-
It is incubated 10 minutes in the 3ml buffer solutions IS of biotin and at 4 DEG C.
2. pass through centrifugal sedimentation cell and abandoning supernatant.
3. repeat step 1 and step 2.
4. cell is resuspended in 10ml buffer solutions IS and is incubated at 25 DEG C to support 10 minutes (for as acceptor knot
Close the dissociation of the CD4 combination Fab fragments of reagent).
5. pass through centrifugal sedimentation cell and abandoning supernatant.
6. step 4 and step 5 are repeated 3 times.
As a result
The principle of reversible Fab- polymers dyeing
The basis of the present invention comes from this surprisingly, it has been found that i.e. receptor binding agent, such as has K to cell receptord<
10-7As little as 10-10High-affinity monomer Fab molecules, it is necessary to by polymerisation agent by multimerization so as to being stably coupled to
Target cell, and targeting rupture that can be through gametophyte C and polymerisation agent binding site Z interphase interaction is complete from cell surface
It is complete to remove (Fig. 1).Further be the discovery that, the key molecule basis of this reversible dyeing be not cell surface receptor molecule with
The compatibility constant K of the interphase interaction of binding site B on receptor binding agentd, but receptor binding agent divides from acceptor
Subsolution from back reaction speed (off rate).Back reaction speed should be 0.5 × 10-4sec-1It is or higher so as to acceptor molecule knot
Closing reagent can remove from target cell completely in rational time window.
In order to be proved to be back reaction speed rather than compatibility is required argument for implementing reversible Fab polymers dyeing
This key is found, is generated following recombinant Fab fragments for cell surface receptor molecule CD4 and (is combined and try as acceptor
Agent)：
1) the Fab fragments being made up of CD4 combination mouse antibody 13B8.2 variable domains and a constant domain, wherein
Constant domain is made up of gamma1 type people light chain constant CH1 domains and kappa type people's light chain constant domains, is such as described in
United States Patent (USP) 7,482,000.The Fab is expressed as 13.B8.2Fab fragments below.
2) mutant 13B8.2Fab fragments, wherein the Phe residue mutations of heavy chain 100 are that (herein also referred to as CD4's Ala dashes forward
Variant 1 (F100A) and with reference to the table II of United States Patent (USP) 7,482,000, entitled F100K-H)
3) mutant 13B8.2Fab fragments, wherein the Tyr residue mutations of light chain 92 are that (herein also referred to as CD4's Ala dashes forward
Variant 2 (Y92A) and with reference to the table III of United States Patent (USP) 7,482,000, entitled Y92L)
4) mutant 13B8.2Fab fragments, wherein the His residue mutations of heavy chain 35 are that (herein also referred to as CD4's Ala dashes forward
Variant 3 (H35A) and with reference to the table II of United States Patent (USP) 7,482,000, entitled H35-H)
5) mutant 13B8.2Fab fragments, wherein the His residue mutations of light chain 91 are that (herein also referred to as CD4's Ala dashes forward
Variant 4 (H91A) and with reference to the table III of United States Patent (USP) 7,482,000, entitled H91-L)
These Fab fragments have wide spectrum compatibility (KdThe scope of value is from 12.5nM to 16.9 μM) and increased Koff- speed
Rate；Summarized and be listed in the table below in 1 by the binding kineticses of BIAcore measure.
1) values listed is the arithmetic mean of instantaneous value for 3 measurements that Bes et al. is provided.
2) Bes et al. provides single measurements.
3) values listed is the arithmetic mean of instantaneous value for 2 measurements that Bes et al. is provided.
For introduce that the heavy chain gene of binding partners C, CD4 combination Fab fragments is fused to arranged in series twoII sequences (SAWSHPQFEK (GGGS)2GGSAWSHPQFEK, as OneSTrEP- sequence labels from Germany
IBA GmbH,It is commercially available), and two chains are all expressed in colibacillus periplasm simultaneously.As acceptor knot
The Fab fragments for closing the functionalization assembling of reagent are then existed by affinity chromatographyPurify, and passing through on resin
After dialysis removes desthiobiotin, in water miscible phycoerythrin mark(PE, make
For polymerisation agent, at least two (about 12) binding site Z, wherein phycoerythrin is provided for One-STrEP- sequence labels
As (fluorescence) mark) in the presence of carry out multimerization.Added Bio (or derivatives thereof, such as such as diaminobiotin
Or desthiobiotin) as competition part after, Streptavidin binding peptide and Streptavidin mutainBetween interaction be reversible.
For dyeing and dissociating experiment, 5 × 106The individual corresponding anti-CD4 Fab- polymers of PBMC use-PE compounds or with corresponding Fab- monomers (the latter not withPE is compound) it is incubated.
Substantially equal outstanding staining signals are all shown after 13B8.2 wild types (wt) Fab fragments and all mutant multimerizations,
And only there is slower KoffSpeed (1.11x10-4s-1And 4.75x10-4s-1) 13B8.2 wt Fab fragments and the energy of mutant 1
It is enough that their antigen (Fig. 2, first row and secondary series) is attached to free state.In the polycomplex of Bio mediation
It is single by adding after (Fig. 2, the 3rd row) rupture and subsequent washingPE (not combining Fab) (Fig. 2,
4th row) come detect the surface of residual combination Fab fragments.As desired by monomer Coloration experiment, mutant 2,3 and 4 does not have
The Fab monomers of residual can be detected, and observe what the cell surface largely remained combined in wild type and the variant of mutant 1
Fab.For mutant 2,3 and 4, cell can use Fab- polymers to mark (Fig. 2, the 5th row) are effectively secondary to dye again,
So as to prove that consistent biotin in the Fab fragment detection process of cell surface residual removes.
Except the analysis (Fig. 2) based on flow cytometry, the complete removal of Fab polymers can also pass through other independent points
Analysis method is assessed, i.e. western blot analysis (Fig. 3).Confirm to be shown in the extremely sensitive FACS results of table 2, mutant 2,
Fab fragments are not all either detected in soluble or insoluble cellular portions after the cracking of 3 and 4 cells, and in solubility
Part can easily detect wild type Fab fragments.Due to this experiment also can detect possibly can not be detected by FACS in
The Fab fragments of change, any significantly internalization for the receptor binding agent that surface combines during dyeing and release can be all excluded,
So as to confirm be shown in Fig. 2 explanation it is correct.With proving that mutant 1 is that the FACS non-fully removed is tested under wash conditions used
On the contrary, the mutant 1 of residual is not detected in Western blot experiment in the cell of similar process, thus it is speculated that be due to albumen
The sensitivity of matter blotting is relatively low.
Main wonderful result is as follows：Due to its low compatibility (KdIt is about 6.2 μM and 16 μM) Fab mutant 3 and 4
In corresponding Fab fragments andPE is as release completely after the rupture of the multivalent complex of polymerisation agent
It is consistent with the teaching in United States Patent (USP) 7,776,562 or international patent application WO02/054065, with monomer wild type Fab molecules
(being similar to mutant 3 and 4Fab fragments) is on the contrary, the complete release of mutant 2Fab fragments is completely unpredictable, because prominent
Compatibility (the K of variant 2d=14.75 ± 2.25nM) it is defined as apparently higher than wild type (Kd=29.17 ± 3.27nM) and it is all
Other mutant.But mutant 2 has faster back reaction speed (k than wild type Fab fragments and Fab mutant 1off=
10.80±1.91x10-4s-1；Also 1) feature is shown in Table, is shown to be the list that dissociation rate rather than compatibility constant combine on surface
There is leading role in the combination stability of body Fab fragments.According to this explanation, all completely reversibility Fab fragment (mutant
One 2-4) is shared (being defined herein as receptor binding agent) and is more than 0.5 × 10-4s-1Fast back reaction speed.But still need to
It should be noted that in the rupture of multivalence combination compound, the long period is washed/is incubated with the solution comprising Bio, then
Wild type Fab fragments and (both K of Fab mutant 1offSpeed is both greater than 0.5 × 10-4s-1) complete in rational time window
Remove what is be possible to, and receptor binding agent therefore can be used as according to the present invention.
In a word, the k that these as shown by data are combined with acceptor molecule via binding site BoffSpeed is more than 0.5 × 10-4s-1
Multimerization receptor binding agent (such as Fab- fragments) can be used for staining cell and these reagents are in life in a stable manner
Can be completely from the surface separation and removal of mark cell under the conditions of reason.
The present invention illustratively described herein may be adapted to lack any key element not specifically disclosed herein or element,
Implement under one or more restrictive conditions.Thus, for example, term "comprising" " comprising " " containing " etc. should be by wide in range and unrestricted
The understanding of property.In addition, terminology employed herein and expression are descriptive rather than restrictive condition, and do not use this art
Language and expression exclude any intention that the equivalent of feature is shown and described or part thereof, but it is to be appreciated that a variety of modifications can
It can be included in the scope of protection of present invention.It is therefore understood that although the present invention by exemplary embodiment and
Optional feature specifically discloses, and technical staff can seek to be embodied in this paper modification and transformation of the invention, these modifications and
Variation is contemplated as falling with the scope of the present invention.
Invention is described widely and generically herein.Each narrower species and to fall into general open scope sub-
Category group all forms the part of the present invention.This includes removing the sheet of the negative limitation of any theme with additional conditions or from the category
Whether the general description of invention, the material no matter removed are the parts of the invention specifically described.
Other embodiments are in following claims.In addition, the feature or aspect of the present invention are in a manner of marlcush group
Description, it would be recognized by those skilled in the art that the present invention can also be with any single member of marlcush group or the shape of member's subgroup
Formula describes.
Claims (10)
1. a kind of method for making the reversible dyeing of target cell using detectable label, the target cell includes acceptor in its surface
Molecule, methods described include：
The cell mixture comprising the target cell is set to be contacted with following：
(i) receptor binding agent, the receptor binding agent includes at least one binding site B, wherein the binding site B is special
The opposite sex is bound to the acceptor molecule, wherein the receptor binding agent via the binding site B and the acceptor molecule it
Between combination dissociation rate constant (koff) value be about 0.5 × 10-4sec-1Or it is bigger, the receptor binding agent enters one
Step include at least one binding partners C, wherein the binding partners C can Reversible binding to polymerisation agent bound site
Point Z；
(ii) polymerisation agent, the polymerisation agent include the reversible of the binding partners C for the receptor binding agent
With reference at least two binding site Z,
Wherein described receptor binding agent (i) and the polymerisation agent (ii) form the multivalence combination for being bound to the target cell
Compound, each multivalence combination compound include at least two acceptors being bound to a polymerisation agent and combined
Reagent, the multivalence combination compound provide increased affinity relative to the receptor binding agent；With
(iii) detectable label, the detectable label are bound to or can be bound to the multivalence combination compound,
Wherein described target cell is dyed by the combination of the multivalence combination compound and the target cell, and wherein through described
The rupture of combination between the binding partners C of receptor binding agent and the binding site Z of the polymerisation agent,
The dyeing of the target cell is reversible.
2. according to the method for claim 1, it is characterised in that the binding site Z and gametophyte C Reversible binding
Dissociation constant (Kd) 10-2M to 10-13In the range of M.
3. according to the method for claim 1, it is characterised in that the acceptor molecule binding reagents and the acceptor molecule it
Between combination dissociation rate constant (koff) value be about 1 × 10-4sec-1Or it is bigger, about 2 × 10-4sec-1Or it is bigger, about 3 ×
10-4sec-1Or it is bigger, about 4 × 10-4sec-1Or it is bigger, about 5 × 10-4sec-1Or it is bigger, about 1 × 10-3sec-1Or it is bigger, about
1.5×10-3sec-1Or it is bigger, about 2 × 10-3sec-1Or it is bigger, about 3 × 10-3sec-1Or it is bigger, about 4 × 10-3sec-1Or more
Greatly, about 5 × 10-3sec-1Or it is bigger, or about 1 × 10-2sec-1It is or bigger.
4. according to the method in any one of claims 1 to 3, it is characterised in that the receptor binding agent with it is described by
Dissociation constant (the K of combination between body moleculed) 10-2M to 10-10In the range of M.
5. according to the method for claim 4, it is characterised in that between the receptor binding agent and the acceptor molecule
With reference to dissociation constant (Kd) in following ranges：About 10-3M to about 10-10M, or about 10-3M to about 10-9M, or about 10-3M is to about
10-8M, or about 10-3M to about 10-7M, or about 10-7M to about 10-10M, or about 10-7M to about 10-9M。
6. method according to any one of claim 1 to 5, it further comprises from being present in the cell mixture
Non-target cell separate the cell of the dyeing.
7. method according to any one of claim 1 to 6, it further comprises by rupturing the receptor binding agent
The binding partners C and the polymerisation agent the binding site Z between combination removed from the target cell
The dyeing.
8. the method according to any one of claim 6 to 7, it is characterised in that the removal of the polymerisation agent causes
Dissociation of the receptor binding agent from the cell of the dyeing.
9. method according to any one of claim 1 to 8, it is characterised in that
(a) the binding partners C includes biotin, and the strepto- that the polymerisation agent includes Reversible binding to biotin is affine
Plain analog or avidin analog；
(b) the binding partners C includes Reversible binding to Streptavidin or the biotin analog of avidin, institute
It is affine to state Streptavidin, avidin, strepto- that polymerisation agent includes Reversible binding to the biotin analog
Plain analog or avidin analog, or
(c) the binding partners C includes Streptavidin or avidin binding peptide, and the polymerisation agent includes can
It is affine against Streptavidin, avidin, the strepto- for being bound to Streptavidin or the avidin binding peptide
Plain analog or avidin analog.
10. according to the method for claim 9, it is characterised in that the binding partners C includes Streptavidin-combination
Peptide Trp-Ser-His-Pro-Gln-Phe-Glu-Lys, the polymerisation agent include Streptavidin analog Va144-
Thr45-Ala46-Arg47Or Streptavidin analog Ile44-Gly45-Ala46-Arg47。
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| US201161508943P | 2011-07-18 | 2011-07-18 | |
| US61/508,943 | 2011-07-18 | ||
| CN201280045377.8A CN103797028B (en) | 2011-07-18 | 2012-07-17 | Make the method for the reversible dyeing of target cell |
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| CN201710565030.0A Active CN107843730B (en) | 2011-07-18 | 2012-07-17 | Method for reversibly staining target cells |
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| US (2) | US9023604B2 (en) |
| EP (2) | EP3418295B1 (en) |
| JP (2) | JP6310388B2 (en) |
| CN (2) | CN103797028B (en) |
| ES (1) | ES2682750T3 (en) |
| HK (1) | HK1258907A1 (en) |
| PL (1) | PL2734538T3 (en) |
| PT (1) | PT2734538T (en) |
| SI (1) | SI2734538T1 (en) |
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| WO (1) | WO2013011011A2 (en) |
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| WO2013011011A3 (en) | 2013-05-02 |
| SI2734538T1 (en) | 2018-09-28 |
| CN103797028A (en) | 2014-05-14 |
| EP2734538B1 (en) | 2018-05-02 |
| TR201811128T4 (en) | 2018-08-27 |
| PT2734538T (en) | 2018-08-02 |
| CN107843730B (en) | 2021-08-20 |
| EP3418295A1 (en) | 2018-12-26 |
| US9023604B2 (en) | 2015-05-05 |
| CN103797028B (en) | 2017-08-25 |
| US20150301046A1 (en) | 2015-10-22 |
| US20140295458A1 (en) | 2014-10-02 |
| US9188589B2 (en) | 2015-11-17 |
| ES2682750T3 (en) | 2018-09-21 |
| JP2018119982A (en) | 2018-08-02 |
| WO2013011011A2 (en) | 2013-01-24 |
| JP6310388B2 (en) | 2018-04-11 |
| PL2734538T3 (en) | 2018-10-31 |
| HK1258907A1 (en) | 2019-11-22 |
| EP2734538A2 (en) | 2014-05-28 |
| JP6770989B2 (en) | 2020-10-21 |
| EP3418295B1 (en) | 2020-09-02 |
| JP2014529361A (en) | 2014-11-06 |
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